BRPI0910461B1 - recombinant follicle stimulating hormone, its pharmaceutical composition, its production method and its use - Google Patents

Abstract

recombinant follicle stimulating hormone (fhs) including sialylation alpha 2,3 and alpha 2,6, their pharmaceutical preparations and compositions, their use in the manufacture of medicines, and the production method of rfsh. the present invention relates to the preparation including recombinant fsh (rfsh).

Description

[001] The present invention relates to gonadotropins for use in the treatment of infertility. In particular, the invention relates to the follicle stimulating hormone (FSH).

[002] Gonadotropins are a group of hormones of heterodimeric glycoproteins that regulate gonadal function in males and females. They include follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG).

[003] FSH is naturally secreted by the anterior pituitary gland and works to support follicular development and evolution. FSH comprises a 92 amino acid alpha subunit, also common to other LH and CG glycoprotein hormones, and a 111 amino acid beta subunit unique to FSH that confers biological specificity of the hormone (Pierce and Parsons, 1981). Each subunit is then translationally modified by the addition of complex carbohydrate residues. Both subunits carry 2 sites for N-linked glycan binding, the alpha subunit at 52 and 78 amino acids and the beta subunit at 7 and 24 amino acid residues (Rathnam and Saxena, 1975, Saxena and Rathnam, 1976). FSH is thus glycosylated at about 30% by weight (Dias and Van Roey. 2001. Fox et al., 2001).

[004] FSH purified from postmenopausal human urine has been used for many years in fertility treatment; both to promote ovulation in natural production and to provide oocytes for assisted reproductive technologies. Two recombinant versions of FSH, Gonal-F (Serono) and Puregon (Organon) became available in the mid-1990s. These are both expressed in hamsterchinese (CHO) ovary cells (Howies, 1996).

[005] There is considerable heterogeneity associated with FSH preparations that refers to differences in the amounts of various isoforms present. The individual FSH isoforms have identical amino acid sequences, but differ in the level at which they are post translationally modified; the particular isoforms are characterized by heterogeneity of the branched carbohydrate structures and different amounts of incorporation of sialic acid (a terminal sugar), both of which appear to influence the bioactivity of the specific isoform.

[006] The glycosylation of natural FSH is highly complex. Osglycans in naturally derived pituitary FSH can contain a wide range of structures that can include combinations of bi-, tri- and tetra-antennary glycans (Pierce and Parsons, 1981. Ryan et al., 1987. Baenziger and Green, 1988). Glycans can also perform modifications: nucleus fucozilation, sectioning glycosamine, extended chains with acetyl lactosamine, partial or complete sialylation, sialylation with α2,3 and α2,6 bonds, south-fatalized egalactosamine replaced by galactose (Dalpathado et al., 2006 ). In addition, there are differences between the distributions of glycogen structures at the individual glycosylation sites. A comparable level of glycan complexity was found in FSH derived from the serum of individuals and the urine of postmenopausal women (Wide et al., 2007).

[007] The glycosylation of recombinant FSH products reflects the range of glycosyl transferases present in the host cell line. The rFSH products are derived from constructed Chinese hamster ovary cells (CHO cells). The range of glycogen modifications in CHO-derived rFSH is more limited than that found in natural products, derived from pituitary or urine extracts. Examples of the reduced glycan heterogeneity found in CHO-derived rFSH include a lack of sectioning glucosamine and a reduced content of core fucolisation and extensions of acetyl lactosamine (Hard et al., 1990). In addition, CHO cells are only able to add sialic acid using α2,3 binding (Kagawa et al., 1988, Takeuchi et al., 1988, Svensson et al., 1990). This is different from the naturally produced FSH that contains glycans with a mixture of sialic acid bound to α2,3 and α2,6.

[008] It has been shown that the preparation of recombinant FSH (Organon) differs in the amounts of FSH with an isoelectric point (pi) below 4 (considering the acidic isoforms) when compared to the FSH of post menopausal urine or serum, pituitary (Ulloa- Aguirre et al., 1995). The amount of acidic isoforms in urinary preparations was much higher when compared to the recombinant products, Gonal-f (Serono) and Puregon (Organon) (Andersen et al., 2004). This should reflect a lower molar content of sialic acid in rFSH since the content of negatively charged sulfate-modified glycan is low in FSH. The lower content of sialic acid, compared to natural FSH, is a characteristic of both commercially available FSH products, and therefore should reflect a limitation in the manufacturing process (Bassett and Driebergen, 2005).

[009] There is a large body of scientific work that analyzes and attempts to explain variations in FSH glycosylation between individuals and changes during the course of an ovulation cycle. One of the main discussions refers to the observation that the concentration of FSH and the content of sialic acid both decrease during the preovulatory phase of the cycle.

[0010] The decreased sialic acid content results in a more basic FSH that is both cleared more quickly and, in at least, is more potent at the target receptor (Zambrano et al., 1996). The question as to the biological relevance of these changes and how they may be involved in the selection of the dominant follicle remains unresolved (reviewed by Ulloa-Aguirre, 2003).

[0011] The circulatory lifespan of FSH has been documented for materials from various sources. Some of these materials were fractionated on the basis of total molecular charge, as characterized by their pi, in which more acid equals a greater negative charge. As previously established, the main contributor to the molecular charge is the total sialic content of each FSH molecule. For example, rFSH (Organon) has a sialic acid content of around 8 mol / mol, while urine-derived FSH has a higher sialic acid content (by Leeuw et al., 1996). The corresponding plasma clearance rates in the rat are 0.34 and 0.14 ml / minute (Ulloa-Aguirre et al., 2003). In another example where a sample of recombinant FSH was divided into high and low pi fractions, the in vivo potency of the high pi fraction (lower sialic acid content) was decreased and had a shorter plasma half-life (D'Antonio and others, 1999). It has also been reported that the most basic FSH circulation during the later stages of the ovulation cycle is due to the sub-regulation of α2,3 sialyl transferase in the anterior pituitary which is caused by increasing levels of estradiol (Damian-Matsumara et al., 1999. Ulloa - Aguirre et al., 2001). The results for α2,6 sialyl transferase have not been reported.

[0012] The total sialic acid content of FSH and rFSH is not directly comparable, since sialic acids are generally linked in two ways. Pituitary / serum / urinary FSH contains sialic acid bound to both α2,3 and α2,6, with a predominance of the former. However, CHO cell-derived combinations only contain α2.3 (Kagawa et al., 1988, Takeuchi et al., 1988, Svens son et al., 1990). This is another difference between natural and current recombinant products in addition to the lower total sialic acid content of the latter.

[0013] CHO cells are generally used for the production of pharmaceutical human recombinant proteins. The structural analysis identified that sialic acid is exclusively bound by an α2,3 bond. (Kagawa et al., 1988, Takeuchi et al., 1988, Svensson et al., 1990). Many glycoproteins contain a mixture of both α2,3 and α2,6 bonds. Consequently, recombinant proteins expressed using the CHO system will differ from their counterparts in their types of bonds. This is an important consideration in the production of biologicals for pharmaceutical use since the carbohydrate portions can contribute to the pharmaceutical attributes of the molecule.

[0014] It is desirable to have an rFSH product that replicates or imitates the physiochemical and pharmacokinetic profile of the product produced from human urine. It is desirable to have an rFSH product that improves the property or pharmacokinetic properties compared to the known combination product.

[0015] In this context according to the present invention recombinant FSH ("rFSH" or "recFSH") is provided including α2,3 sialylation and α2,6 sialylation and, optionally α2,8 sialylation. The rFSH (or rFSH preparation) according to the invention can have 10% or more of the total sialylation being α2,3 sialylation, for example 65 to 85% of the total sialylation can be α2,3 sialylation. The rFSH (or rFSH preparation) of the invention can have 50% or less of the total sialylation being sialylation α2,6, for example, 15 to 35% of the total sialylation can be sialylation α2,6. The rFSH (or rFSH preparation) of the invention can have 5% or less of the total sialylation being sialylation α2.8, for example 0.1 to 4% of the total sialylation can be siaylation α2.8. The rFSH (or preparation of rFSH) according to the invention can have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 moles / moles or greater, for example between 6 moles / moles and 15 moles / moles.

[0016] Applicants have found that the type of binding of sialic acids, α2,3- or α2,6-, can have a dramatic influence on the biological clearance of FSH. Human cell lines, as opposed to CHO cell lines, can express recombinant FSH with sialic acids linked by both α2,3 and α2,6 bonds. In Example 4 a recombinant FSH cell line was made, which the expressed FSH containing glycans with low levels of both sialic acid bound to α2,3 and α2,6 (figure 6). This basic material, with limited sialic acid content (figure 4) was cleared very quickly from the rat circulation as expected (figure 7). A cell line was then subjected to a second engineering step with the addition of the gene coding for α2,6-sialyl transferase (Example 5). The resulting rFSH was highly sialylated showing sialic acid content and pi distribution comparable to urinary FSH (figure 5). However, the material was cleared very quickly from the circulation of rats at a rate comparable to the original material, which had a low sialic acid content (figure 8). This was an unexpected observation since it is known that a proportion of sialic acid in natural and biologically active FSH is linked by α2,6. The clearance of α2,6 sialylated rFSH was found to be mediated by the asialoglycoprotein receptor (ASGP) found in the liver (Example 9). This was demonstrated by transient blocking of ASGP receptors using an excess of another substrate for the receptor. With the receptor blocked by asialofetuin, the expected clearance for the highly sialylated material was restored (figure 9). This was maintained for several hours until the blockade was overcome and the highly sialylated rFSH linked to α2,6 summarized its rapid clearance.

[0017] The recombinant FSH with a mixture of sialic acid bound to both α2,3 and α2,6 was made by building a human cell line to express both rFSH and c-2,3 sialyltransphere (Examples 4 and 5 ). The expressed product is highly acidic and carries a mixture of sialic acids both linked to α2,3- and α2,6; the latter provided by endogenous sialyltransferase activity (figure 6). This has two advantages over rFSH expressed in conventional CHO cells: first, the material is more highly sialylated due to the combined activities of the two sialyltransferases; and secondly, the material most closely resembles the natural FSH. This should probably be more biologically appropriate compared to recombinant products derived from CHO cells that produce only α2,3-bound sialic acid (Kagawa et al., 1988, Takeuchi et al., 1988, Svensson et al., 1990) and have an acid content decreased sialic (Ulloa-Aguirre et al., 1995., Andersen et al., 2004).

[0018] Applicants have surprisingly found that the orFSH of the invention can more closely replicate or imitate the physiochemical and pharmacokinetic profile of the natural human urinary product than other recombinant products. In other words, the invention's rFSH may be closer to the "natural" FSH. This can have significant advantages with respect to dosage etc. In addition, a "natural" or more "human" product may be more desirable for the patient, who may want therapy, albeit in an artificial sense, to be as "natural" as possible. There may be other advantages (for example, pharmacokinetic advantages) in a recombinant product having a carbohydrate structure (for example, glycan) that is closer to natural FSH (for example, human urinary) than other recombinant products.

[0019] The invention is thus a recombinant version of FSH that carries a mixture of sialic acid of α2,3 and α2,6 and therefore more closely resembles natural FSH. It is expected that the use of this compound for controlled ovarian stimulation, in IVF techniques, and ovulation induction will result in a more natural stimulation of the ovary compared to existing recombinant products.

[0020] According to the present invention in this context FSH ("rFSH" or "recFSH") (and / or a combined FSH preparation) is provided including α2, 3 sialylation and α2,6 sialylation. The rFSH or rFSH preparation can optionally also include α2, 8 sialylation.

[0021] Here, the term "preparation of recombinant FSH" included a preparation, for example, pharmaceutical use which includes FSH recombinant. In the embodiments of the invention, rFSH can be present as a single isoform or as a mixture of isoforms.

[0022] The rFSH (or rFSH preparation) according to the invention can have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 moles / moles or greater ( Example 8), for example between 6 moles / moles and 15 moles / moles, for example, between 8 moles / moles and 14 moles / moles, for example between 10 moles / moles and 14 moles / moles, for example, between 1 moles / moles and 14 moles / moles, for example, between 12 moles / moles and 14 moles / moles, for example, between 12 moles / moles and 13 moles / moles. The rFSH of the invention can be produced or expressed in a human cell line.

[0023] The rFSH (or preparation of rFSH) according to the invention can have 10% or more of the total sialylation being sialylation α2,3. For example, 20, 30, 40, 50, 60, 70, 80 or 90% or more of total sialylation can be α2,3 sialylation. RFSH (or rFSH preparation) can include α2,3 sialylation in an amount that is 65 to 85% of total sialylation, for example 70 to 80% of total sialylation, for example from 71 to 79% of total sialylation . The rFSH (or rFSH preparation) of the invention can have 50% or less of the total sialylation being sialylation α2,6. For example, 40, 30, 20, 10, 5% or less of total sialylation can be α2,6 sialylation. RFSH (or rFSH preparation) can include a2,6-sialylation in an amount that is 15 to 35% of total sialylation, for example 20 to 30% of total sialylation, for example 21 to 29% of total sialylation . The rFSH (or rFSH preparation) of the invention can have 5% or less of the total sialylation being sialylation α2,8. For example, 2.5% or less of total sialylation may be α2.8 sialylation. The rFSH (or rFSH preparation) can include α2,8 sialylation in an amount that is 0.1 to 4% of total sialylation, for example from 0.5 to 3% of total sialylation, for example from 0.5 to 2.5% of total sialylation, sialylation means the amount of sialic residues present on the FSH carbohydrate structures. Α2,3 sialylation means sialylation at position 2,3 (as is well known in the art) and α2,6 sialylation at position 2,6 (also well known in the art). Thus, "% of total sialylation can be α2,3 sialylation" refers to% of the total number of sialic acid residues present in FSH that are sialylated at position 2.3. The term "% of total sialylation being α2,6-sialylation" refers to% of the total number of sialic acid residues present in FSH that are sialylated at position 2,6.

[0024] The rFSH (or rFSH preparation) according to the invention may have a sialic acid content (amount of sialylation per FSH molecule) (based on the mass of protein, instead of the mass of protein plus carbohydrate) of 6% or more (for example between 6% and 15%, for example, between 7% and 13%, for example, between 8% and 12%, for example, between 11% and 15%, for example, between 12% and 14%) by mass.

[0025] Recombinant FSH expressed in hamsterchinese ovary (CHO) cells exclusively includes α2,3 sialylation (Kagawa et al., 1988, Takeuchi et al., 1988, Svensson et al., 1990).

[0026] The rFSH of the invention can be produced or expressed in a human cell line. This can simplify (and yield greater efficiency) the production method, because the manipulation and control, for example, of the cell growth medium to retain sialylation can be less critical than with known processes. The method may also be more efficient because there is little basic rFSH produced than in other production of known rFSH products; More acidic rFSH is produced and the separation / removal of basic FSH is less of a problem. RFSH can be produced or expressed in a Per.C6 cell line, a Per.C6-derived cell line or a modified Per.C6 cell line. The cell line can be modified using α2,3-sialyltransferase. The cell line can be modified using α2,6-sialyltransferase. Alternatively or in addition, rFSH may include sialic acids bound to α2,6 (sialylation α2,6) provided by endogenous [cell line] sialyltransferase activity.

[0027] rFSH can be produced using α2,3- and / or α2,6- sialyl transferase. RFSH can be produced using α2,3-sialyltransferase. RFSH can include sialic acids linked to α2,6 (α2,6 sialylation) supplied by endogenous sialyltransferase activity.

[0028] According to the present invention in another aspect a method of producing rFSH and / or a preparation of rFSH is provided as described here (according to the aspects of the invention) comprising the step of producing or expressing rFSH in a strain human cell, for example a Per.C6 cell line, a Per.C6-derived cell line or a modified Per.C6 cell line, for example a cell line that has been modified using α2,3-sialyltransferase.

[0029] The rFSH structure contains portions of glycan. Branching can occur with the result that the glycan can have 1, 2, 3, 4 or more terminals of sugar residues or "antennae", as is well known in the art. The rFSH of the invention can have glycans with the presence of sialylation in monoantennary and / or diantenary and / or triennialtenary and / or tetra-antennary structures. The rFSH can preferably include monosylylated, disialylated, trisiallylated and tetrasylylated glycan structures with relative amounts as follows: 9 to 15% monoarylated; 2 to 30% dialylylated; 30 to 36% trisiallylated and 25 to 29% tetrasialylated (for example as shown by WAX analysis of charged glycans, as set forth here in Example 8 c).

[0030] According to the present invention in another aspect rFSH produced (e.g., expressed) in a human cell line is provided. The rFSH can include sialylation α2,3 and α2,6. RFSH can be produced or expressed in a Per.C6 cell line, a Per.C6-derived cell line or a modified Per.C6 cell line. The cell line can be modified using α2,3-sialyltransferase. The cell line can be modified using α2,6-sialyltransferase. Alternatively or in addition, rFSH may include sialic acids bound to α2,6 (sialylation α2,6) provided by endogenous [cell line] sialyltransferase activity. The rFSH (or preparation of rFSH) can have 10% or more of the total sialylation sendosylylation α2,3, for example, 65 to 85% of the total sialylation can be sialation α2,3. The rFSH (or rFSH preparation) of the invention can have 50% or less of the total sialylation being a2,6-sialylation, for example, 15 to 35% of the total sialylation can be sialylation α2,6. The rFSH (or rFSH preparation) of the invention can have 5% or less of the total sialylation being sialylation α2.8, for example 0.5 to 4% of the total sialylation can be siaylation α2.8. The rFSH can have a sialic acid content [expressed in terms of a mole ratio of sialic acid to protein moieties] of 6 moles / moles or greater, for example between 6 moles / moles and 15 moles / moles.

[0031] According to the present invention in another aspect a pharmaceutical composition comprising rFSH including α2,3 sialylation and a2,6-sialylation sialylation (for example, as set out above) is provided. The pharmaceutical composition can also comprise hCG and / or LH.

[0032] hCG can be obtained by any means known in the art. hCG as used herein includes recombinant and human derived hCG. Human-derived hCH can be purified from any appropriate source (e.g., urine, and placenta) by any method known in the art. Methods of expression and purification of recombinant hCG are well known in the art.

[0033] LH can be obtained by any means known in the art. LH, as used herein, includes recombinant and human-derived LH. Human-derived LH can be purified from any appropriate source (e.g., urine) by any method known in the art. Methods of expression and purification of LH are well known in the art.

[0034] The pharmaceutical composition can be for the treatment of infertility, for example, for use in, for example, assisted reproductive technologies (ART), ovulation induction or intrauterine insemination (IUI). The pharmaceutical composition can be used, for example, in medical indications where FSH preparations are used. The present invention also provides for the use of rFSH and / or an rFSH preparation described here (in accordance with aspects of the invention) for, or in the manufacture of, a medicament for the treatment of infertility. The pharmaceutical compositions of the present invention can be formulated into compositions well known for any drug delivery routine, for example, oral, rectal, parental, transdermal (e.g., plaster technology), intravenous, intramuscular, subcutaneous, intrasusternal, intravaginal, intraperitoneal, local (powders, ointments or drops) or as a mouth or nasal spray. A typical composition comprises a pharmaceutically acceptable carrier, such as aqueous solution, non-toxic excipients, including salts and preservatives, buffers and the like, as described in Remington's Pharmaceutical Sciences fifteenth edition (Matt Publishing Company, 1975), on pages 1405 to 1412 and 1461 - 87, and in the national formulary XIV fifteenth edition (American Pharmaceutical Association, 1975), among others.

[0035] Examples of suitable aqueous and non-aqueous pharmaceutical vehicles, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as such as olive oil), and injectable organic esters such as ethyl oleate.

[0036] The compositions of the present invention can also contain additives such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Antibacterial and antifungal agents can be included to prevent microbial growth and include, for example, paraben, chlorobutolol, phenol, sorbic acid, and the like. In addition, it may be desirable to include isotonic agents such as sugars, sodium chloride, and the like.

[0037] In some cases, for prolonged effect it is desirable to decrease the absorption of FSH (and other active ingredients, if present) by subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of FSH then its rate of dissolution which, in turn, may depend on the crystal size and crystalline form. Alternatively, the delayed absorption of a parenterally administered form of FSH combination is accomplished by dissolving or suspending the FSH combination in an oily vehicle.

[0038] Injectable deposit forms can be made by forming microencapsulated matrices of FSH (and other agents, if present) in biodegradable polymers such as polyacid-polyglycolide. Depending on the ratio of FSH to polymer and the nature of the particular polymer employed, the rate of FSH release can be controlled. Examples of other biodegradable polymers include polyvinylpyrrolidone, poly (orthoesters), poly (anhydrides) etc. Injectable depot formulations are also prepared by capturing FSH in liposomes or microemulsions that are compatible with body tissues.

[0039] Injectable formulations can be sterilized, for example, by filtration through a bacteria retention filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium just before use. Injectable formulations can be supplied in any suitable container, for example, vial, pre-filled syringe, injection cartridges, and the like.

[0040] Injectable formulations can be supplied as a product having pharmaceutical compositions containing FSH (optionally with hCG, LH etc.) if there is more than one active ingredient (i.e., FSH and, for example, hCG or LH), this can be suitable for separate or joint administration. If administered separately, administration can be sequential. The product can be supplied in any suitable packaging. For example, a product may contain several pre-filled syringes containing FSH, hCG, or a combination of both FSH and hCG, syringes packaged in a blister pack or other means to maintain sterility. A product can optionally contain instructions for using FSH and hCG formulations.

[0041] The pH and concentration of the various components of the pharmaceutical composition are adjusted according to routine practice in this field. See GOODMAN and GILMAN's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICES, 7th edition. In a preferred embodiment, the compositions of the invention are provided as compositions for parenteral administration. General methods for preparing parenteral formulations are known in the art and are described in REMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY, supra, on pages 780 to 820. Parenteral compositions can be supplied in liquid formulation or as a solid that will be mixed with a sterile injectable medium just prior to administration. In an especially preferred embodiment, parenteral compositions are provided in unit dosage form for ease of administration and uniformity of dosage.

Detailed Description of the Invention

[0042] The present invention will now be described in more detail with reference to the following examples and the following drawings in which: Figure 1 shows a plasmid map of the pFSHalfa / beta expression vector; Figure 2 shows the expression vector of α2,3-sialyltransferase (ST3GAL4); Figure 3 shows the expression vector of α2,6-sialyltransferase (ST6GAL1); Figure 4 shows isoelectric focus of recombinant FSH produced by Per.C6 cells stably expressing FSH; Figure 5 shows an example of clones analyzed by isoelectric focusing of recombinant FSH produced by Per.C6 cells stably expressing FSH after engineering with α2,3- or α2,6-sialyltransferase; Figure 6 shows analysis of the Per.C6 FSH sialic acid bonds; figure 7 shows the metabolic clearance rates (MCRs) of FS.C samples from Per.C6; Figure 8 shows MCRs from Per.C6 FSH samples made by α2,6-sialyltransferase; Figure 9 shows MCRs from Per.C6 FSH samples made by α2,6-sialyltransferase; Figure 10 shows MCRs from Per.C6 FSH samples constructed by α2,3-sialyltransferase; Figure 11 shows an increase in ovarian weight by Per.C6 rFSH clones of parental Per.C6 rFSH, according to the method of Steelman and Pohley (1953); Figure 12 shows an increase in ovarian weight by Per.C6 rFSH clones of Per.C6 rFSH constructed (α2,6-sialyltransferase); and figure 13 shows an increase in ovarian weight by Per.C6 rSFH clones of Per.C6 rFSH constructed (α2,3-sialyltransferase).

Sequence Selection Human FSH

[0043] The gene coding region for FSH alpha peptide was used according to Fiddes and Goodman. (1981). The sequence is deposited as AH007338 and at the time of construction there was no variant of this protein sequence. The sequence is referred to here as SEQ ID 1.

[0044] The gene coding region for FSH beta polypeptide was used according to Keene et al. (1989). The sequence is deposited as NM_000510 and at construction time, no other variant of this protein sequence is heard. The sequence is referred to here as SEQ ID 2.

Sialyltransferase

[0045] α2,3-sialyltransferase - The coding region of the parabeta-galactoside alpha-2,3-sialyltransferase 4 gene (α2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is deposited as L23767 and referred to here as SEQ ID 3.

[0046] α2,6-Sialyltransferase - The gene coding region for beta-galactosamide alpha-2,6-sialyltransferase 1 (α2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al. (1990). The sequence is deposited as NM_003032 and referred to here as SEQ ID 4.

EXAMPLES Example 1: Construction of the FSH expression vector

[0047] The coding sequences of FSH alpha polypeptide (AH007338, SEQ ID 1) and FSH beta polypeptide (NM_003032, SEQ ID 2) were amplified by PCR using the starter compositions FSHa-fw and FSHa-rev and FSHb-fw and FSHb -rec respectively. FSHa-fw 5’-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3 'FSHa-retf 5’-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3 ‘FSHb-fw 5-CCAGGCGCGCCACCATGAGGACTACTGA

The resulting amplified FSH beta DNA was digested with Ascl and Hpal restriction enzymes and inserted into the Ascl and Hpal sites on the CMV-regulated mammalian expression vector carrying a neomycin selection marker. Similarly, the FSH alpha DNA was digested with BamHI and Nhel and inserted into the BamHI and Nhel sites on the expression vector already containing the FSH beta polypeptide DNA.

[0049] The DNA vector was used to transform the E.coli strain of DH5α. Sixty clones were captured for amplification and fifty-seven contained the vector containing both FSH alpha and beta. Twenty of these were selected for sequencing and all contained the correct sequencing according to SEQ ID 1 and SEQ ID 2. Plasmid pFSH A + B # 17 was selected for transfection (figure 1).

Example 2: construction of the ST3 expression vector

The alpha-2,3-sialyltransferase 4 beta-galactoside coding sequence (ST3, L23767, SEQ ID 3) was amplified by PCR using the 2.3STfw and 2.3STrev primer combination. 2.3STfw 5’-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC 3 ’2,3STrev S-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-y

The resulting amplified ST3 DNA was digested with restriction enzymes BamHI and AFlll and inserted into the SamHI and AfIll sites on the CMV-regulated mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST3 # 1 (figure 2) contained the correct sequence according to SEQ ID 3 and was selected for transfection.

Example 3: construction of the ST6 expression vector

The beta-galactosamide alpha-2,6-sialyltransferase 1 coding sequence (ST6, NM_003032, SEQ ID 4) was amplified by PCR using the 2.6STfw and 2.6STrev primer combination. 2,6STfw 5, -CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3, 2,6STrev S -TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-S ''

The resulting amplified ST6 DNA was digested with the restriction enzymes SamHI and AFlll and inserted into the SamHl and AFlll sites on the CMV-regulated mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST6 # 11 (figure 3) contained the correct sequence according to SEQ ID 4 and was selected for transfection.

Example 4: stable expression of pFSH A + B in PER.C6 cells. Isolation and analysis of transfection of clones.

[0054] Per.C6 clones producing FSH were generated by expression of both FSH polypeptide chains from a single plasmid (see Example 1).

[0055] To obtain stable clones a liposome based on the transfection agent with the construction of pFSH A + B. Stable clones were selected in VPRO supplemented with 10% FCS and containing G418. In the weeks after transfection, G418 resistant clones developed. A total of 250 clones were selected for isolation. The isolated clones were cultured in a selection medium up to 70 to 80% of confluents. Supernatants were assayed for FSH protein content using a selective FSH ELISA and pharmacological activity at the FSH receptor in cloned cell line, using a cAMP accumulation assay. Clones (98) expressing functional protein were progressed to culture expansion in 24-well, 6-well and T80 flasks.

[0056] Studies to obtain material productivity and quality from seven clones were started in T80 flasks to generate enough material. The cells were grown in supplemented medium as previously described for 7 days and the supernatant harvested. Productivity was determined using the FSH selective ELISA. The isoelectric profile of the material was determined (Example 6). Representative samples are shown in figure 4. IEF information was used to select clones for analysis of metabolic clearance rate (Example 9). Clones (005, 104, 179, 223, 144) with sufficient productivity and quality were selected for syllabyltransferase engineering.

Example 5: Sialylation level is increased in cells that overexpress α2,3- or α2,6-sialyltransferase. Stable pST3 or pST6 expression in PER.C6 FSH expression cells; Isolation and analysis of transfection of clones.

[0057] Per.C6 clones producing highly sialylated FSH were generated by expression of α2,3 sialyltransferase or α2,6 sialyl transferase from separate plasmids (see examples 2 and 3) in PER.C6 cells previously expressing both strands of FSH polypeptide (see Example 4). Four clones produced from PER.C6® cells as set forth here in Example 4 were selected for their characteristics including productivity, good growth profile, production of functional protein, and FSH produced that included some sialylation.

[0058] Stable clones were generated as previously described in Example 4. A total of 202 clones from the α2,3-sialyltransferase program and 210 clones from the α2,6-sialyltransferase program were isolated, expanded and tested. The final clone number for the α2.3 study was 12 and 30 for the α2.6 study.

[0059] The α2,3-sialyltransferase clones were adapted to serum-free media and suspension conditions.

[0060] As before, the clones were assayed using an FSH selective ELISA, functional response in an FSH receptor cell line, IEF (Example 6), metabolic clearance rate (Example 9) and Steelman Pohley analysis (Example 10). The results were compared to a commercially available recombinant FSH (Goalal-f, Serono) and the parenteral Per.C6 FSH cell lines. Representative samples are known in figure 5. Some clones do not show an increase in sialylation, but it can be seen that the FSH produced by most clones has significantly improved sialation (ie, on average, more FSH isoforms with high numbers of sialic acids) compared with FSH expressed without α2,3- or α2,6- sialyltransferase.

[0061] In conclusion, the expression of FSH along with sialyltransferase in PER.C6 cells results in increased levels of sialylated FSH compared to cells expressing FSH only.

Example 6: Pi analysis of FSH isoforms produced by Per.C6 by isoelectric focusing.

[0062] Electrophoresis is defined as the transport of charged molecules by means of a solvent through an electric field. The mobility of a biological molecule through an electric field will depend on the intensity of the field, net charge on the molecule, shape size of the molecule, ionic intensity and properties of the medium through which the molecules migrate.

[0063] Isoelectric focusing (IEF) is an electrophoretic technique for separating proteins based on their pi. Pi is the pH at which a protein has no net charge and will not migrate in an electric field. The sialic acid content of the FSH isoforms subtly alters the pi point for each isoform, which can be explored using this technique to visualize the Per.C6 FSH isoforms of each clone.

[0064] The isoelectric points of the FSH isoforms produced by Per.C6 in cell culture supernatants were analyzed using isoelectric focusing. Per.C6 FSH clone cell culture medium was produced as described in examples 4 and 5.

[0065] Per.C6 FSH samples were separated in Novex® IEFGels containing 5% polyacrylamide under negative conditions in a gradient of pH 3.0 to 7.0 in a pH 3.0 to 7.0 of ampholyte solution.

[0066] Proteins were transferred onto sustained nitrocellulose and visualized using a primary anti-FSHα monoclonal antibody, secondary anti-ammonium IgG alkaline phosphate antibody and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitrazul tetrazolium reagent (NBT) to visualize the bandages.

[0067] As indicated in figures 4 and 5, the bandages represent isoforms of FSH containing different numbers of sialic acid molecules.

[0068] Using this method, clones producing FSH isoforms with a greater number of sialic acid molecules have been identified. Engineering with α2,3- or α2,6- sialyltransferase resulted in clones with more sialic acid and less.

Example 7: Analysis of Per.C6 FSH sialic acid bonds

[0069] Glycoconjugates were analyzed using a lectin-based glycan differentiation method. With this method, glycoproteins and glycoconjugates linked to nitrocellulose can be characterized. Lectins selectively recognize a particular portion, for example, sialic acid bound to α2,3. The applied lectins are conjugated with the steroid hapten digoxigenin which allows the immunological detection of bound lectins.

[0070] The Per.C6 FSH purified from a parental clone (no additional sialyltransferase), a clone constructed by α2,3-sialyltransferase and a clone constructed by α2,6-sialyltransferase were separated using standard SDS-PAGE techniques. A commercially available recombinant FSH (Gonal-f, Serono) was used as a standard.

[0071] Sialic acid was analyzed using the DIG Glycan Differentiationkit (Cat. N ° 11 210 238 001, Roche) according to the manufacturers' instructions. Positive reactions with Sambucus nigra agglutinin (SNA) indicated terminally bound sialic acid (α2-6). Positive reactions with Maackia amurensis Il (MAA) agglutinin indicated terminally bound sialic acid (α2-3).

[0072] In summary, parental clone 005 contained low levels of both sialic acid of α2,3 and α2,6. Clones constructed with α2,3-sialyltransferase contained high levels of α2,3 sialic acid bonds and low levels of α2,6 sialic acid bonds. Clones constructed with α2,6-sialyltransferase contained high levels of α2,6 sialic acid bonds and low levels of α2,3 sialic acid bonds. The standard Gonal-f control contains only α2,3 sialic acid bonds. This is consistent with what is known about recombinant proteins produced in Chinese hamster ovarian cells (Kagawa et al., 1988, Takeuchi et al., 1988, Svensson et al., 1990).

In conclusion, engineering Per.C6 FSH cells with α2,3- or α2,6- sialyltransferase successively increased the number of sialic acid molecules conjugated to FSH in the samples.

Example 8a: Quantification of total sialic acid

[0074] Sialic acid is a protein-bound carbohydrate considered to be a monosaccharide and occurs in combination with other monosaccharides such as galactose, mannose, glucosamine, galactosamine and fucose.

[0075] Total sialic acid in purified rFSH (Example 11) was measured using an enzymatic sialic acid quantification kit according to the manufacturers' protocol (Sigma, Sialic-Q). In summary, N-acetylneuraminic acid aldolase catalyzes sialic acid to N-acetylmannasine and pyruvic acid. Pyruvic acid can be reduced to lactic acid by β-NADH and lactic dehydrogenation. The oxidation of B-NADH can be accurately measured spectrophotometrically.

[0076] Protein concentration was measured on microtiter plates using a commercial bicinconamic acid (BCA) assay kit (Sigma, B 9643) based on the Lowry method (Lowry et al., 1951).

[0077] The total FSH sialic acid content of Per.C6 was measured and found to be greater than 6 mol / mol.

Example 8b: Quantification of relative amounts of sialic acid from α2,3, α2,6 and α2,8.

[0078] The relative percent amounts of sialic acid of α2,3, α2,6 and α2,8 in purified rFSH (Example 11) were measured using known techniques.

[0079] Each rFSH sample was immobilized (gel block), washed, reduced, alkylated and digested with PNGase F overnight. The N-glycans were then extracted and processed. N-glycans for NP-HPLC and WAX-HPLC analysis were labeled with fluorophore 2AB as detailed in Royle et al. The N-glycans were worked on normal phase HPLC (NP) on a TSK amide column (as detailed in Royle et al.) With retention time expressed in glucose units (GU).

[0080] The samples extracted, mixed, glycans (extracted as above) were digested with different sialidases to determine the bonds. NAN 1 (recombinant sialidase) releases non-reducing terminal sialic acids linked to α2,3 (NeuNAc and NeuNGc), ABS (Arthrobacter ureafaciens sialidase) releases non-reducing terminating sialic acids linked to α2,3, α2, 6 and α2.8 (NeuNAc and NeuNGc). The samples were analyzed by NP-HPLC, to allow comparison of the undigested sample with that digested with NAN1 and that digested with ABS. The comparison of these three traces of NP-HPLC (undigested, digested with NAN1, digested with ABS) shows that digestion with ABS and NAN1 provides different results. This indicates that the samples have sialic acids with α2,3, α2,6 and α2,8 bonds. The relative percentages were calculated from structures present in undigested glycan mixtures and were found to be in the ranges of 65% to 85% (for example, 77.75%) for α2,3 sialylation; 15 to 35% (for example, 21.46%) for α2.6 sialylation; and 0.1 to 3% for α2.8 sialylation.

Example 8c: Quantification of relative amounts of sialylated mono-, di-, tri- and tetra antennary structures

[0081] The relative percentage amounts of sialylated mono, di, tri and tetra structures in glycans extracted from purified rFSH (Example 11) were measured using known techniques.

[0082] Each rFSH sample was immobilized (gel block), washed, reduced, alkylated and digested with PNGase F overnight. The N-glycans were then extracted and processed. N-glycans for NP-HPLC and WAX-HPLC analysis were labeled with fluorophore 2AB as detailed in Royle et al.

[0083] Weak anion exchange HPLC (WAX) to separate N-glycans per charge (Example 8b) was performed as established here at Royle et al., With a fetuin N-glycan standard as a reference. Glycans were eluted according to the number of sialic acids they contained. All samples included mono (1S), di (2S), tri (3S) and sialylated tetra (4S) structures. The relative amounts of sialylated structures have been found to be in the following ratios (1S: 2S: 4S: 4S): 9 to 15%: 27 to 30%: 30 to 36%: 25 to 29% (for example, 10.24: 28 , 65: 35,49: 25,62).

Example 9: Determination of metabolic clearance rates of rFSH

[0084] To determine the metabolic clearance rate (MCR) of FSH from Per.C6 samples, conscious female rats (3 three animals per clone) were injected into the tail vein at time zero with an rFSH bolus (1 - 10 μg / rat, based on ELISA quantification of samples, DRG EIA 1288). Blood samples (400 μl) were collected from the tip of the tail at 1, 2, 4, 8, 12, 24 and 32 hours after the injection of the test sample. Serum was collected by centrifugation and tested for FSH content by ELISA (DRG EIA 1288).

[0085] The asialoglycoprotein receptor (ASGP-R) recognizes desiallylated (galactose-terminated) glycoproteins such as asia-lofetuin (ASF). (Pricer and Ashwell, 1971. Van Lenten and Ashwell, 1972). The ASGP receptor and the linked desialylated glycoproteins are internalized in the cell where the receptor is recycled and the ligand is degraded (Regoeczi et al., 1978, Steer and Ashwell, 1980).

[0086] To investigate whether the Per.C6 FSH material was purified using this mechanism, ASGP-R saturated with asialofetuin. The metabolic clearance rate of α2,6 or α2,3-sialyltransferase, parental material was determined as described with co-administration of a 7500-fold minimal molar excess of asialofetuine to saturate ASGP-R for 1 to 2 hours .

[0087] The material produced by the parental Per.C6 FSH clones contained slightly more durable MCR material, but a high percentage was cleared quickly (figure 7). The lead clone that contained 005 that contained more sialylated material was constructed using α2,6- or α2,3-sialyltransferase (Example 5). Although the clones constructed with α2,6-sialyltransferase demonstrated increased sialylation (figure 5) there was no improvement in the MCR (figure 7). The ASGR block restored the MCR of the α2.6 material to that of the standard, demonstrating that, even with increased α2.6 bonds, the material is cleared quickly (figure 8). Engineering with α2,3-sialyltransferase resulted in clones with MCR comparable to the standard (figure 9) and variation in sialic content was consistent with that known for FSH isoforms (figure 10).

Example 10: Steelman-Pohley in vivo assay

[0088] To demonstrate increased sialic acid content over FSH results in an increased biological effect, the increase in ovarian weights in rats by highly sialylated FSH as produced in Example 5 was examined.

[0089] The increase in ovarian weights due to Per.C6 rFSH clones were analyzed according to the method of Steelman and Pohley (1953). The Per.C6 rFSH from filtered cell media samples was quantified by ELISA (DRG1EIA-1288). The samples (Per.C6 rFSH) and standards (Gonal-f rFSH) were tested in five different doses (3 animals / dose). Gonal-f was dosed at 50, 100, 200, 400, and 800 ng / rat. Sample doses were calculated using their AUC values relative to Gonal-f, typically 0.05 to 10 μg / rat.

[0090] In conclusion, the subsialylated material produced by the parental Per.C6 FSH clones (figure 11) was not as potent in the ovarian weight gain assay as the commercially available rFSH. Sialyltransferase engineering to add additional α2.6 bonds high in sialic acid content but did not provide potency in the in vivo assay (figure 12).

[0091] However, the additional α2.3 bonds significantly improved the potency (figure 13) and the two FSH preparations in combination (Per.C6 and CHO derivative) showed very similar profiles in this assay.

Example 11: Evaluation of production and purification

[0092] The procedure was developed to produce FSH in PER.C6 cells that were cultured in suspension in serum-free medium. The procedure is described below and has been applied to several FSH-producing PER.C6 cell lines.

[0093] The FSH of parental clone 005, clone α2,3 007 and clone α2,6059 were prepared using a modification of the method described by Lowry et al. (1976).

[0094] For the production of PER.C6-FSH, the cell lines were adapted to a serum-free medium, that is, Excell 525 (JRH Biosciences). The cells were first cultured to form a 70% to 90% confluent monolayer in a T80 culture flask. In passing, the cells were resuspended in serum-free medium, Excell 525 + 4 mM L-Glutamine, at a cell density of 0.3 x 10 6 cells / ml. A 25 ml cell suspension was placed in a 250 ml shaker bottle and stirred at 100 rpm at 37 ° C in 5% CO2. After reaching a cell density of> 1X106 cells / ml, the cells were underdeveloped to a cell density of 0.2 or 0.3x106 cells / ml and also grown in shaker flasks at 37 ° C, 5% CO2 and 100 rpm.

[0095] For the production of FSH, the cells were transferred to a serum-free production medium, that is, VPRO (JRH Biosciences), which supports the growth of PER.C6 cells at very high cell densities (usually cells / ml> 107 in a batch culture). The cells were first cultured at cells / ml> 1x106 in Excell 525, then spun for 5 minutes at 1000 rpm and subsequently suspended in 6 mM VPRO + L-glutamine at a density of 1x106 cells / ml. The cells were then cultured in a shaker flask for 7 to 10 days at 37 ° C, 5% CO2 and 100 rpm. During this period, the cells grew to a density of> 107 cells / ml. The culture medium was harvested after cell viability started to decline. The cells were spun for 5 minutes at 1000 rpm and the supernatant was used for FSH quantification and purification. The FSH concentration was determined using ELISA (DRG EIA 1288).

[0096] Thereafter, FSH purification was performed using a modification of the method described by Lowry and another (1976). I was obtained by DEAE cellulose chromatography, Sephadex G100 gel filtration, hydroxyapatite absorption chromatography, and preparative polyacrylamide electrophoresis.

[0097] During all chromatographic procedures, the presence of immunoreactive FSH was confirmed by RIA (DRG EIA 1288) and IEF (Example 6).

References

[0098] Andersen CY, Westergaard LG, and van WeIy M. (2004) .FSH isoform composition of commercial gonadotrophin preparations: a neglected aspect? Reprod Biomed Online. 9 (2), 231-236.

[0099] Arey BJ, Stevis PE, Deecher DC, Shen ES, Frail DE, Ne-gro-Vilar A, and Lopez FJ. (1997) Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms. MoI Endocrinol. 11 (5), 517-526.

[00100] Baenziger JU and Green ED. (1988). Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim Biophys Acta. 947 (2), 287-306.

[00101] Bassett RM, and Driebergen R. (2005). Continued improvements in the quality and consistency of follitropin alpha, recombinant human FSH. Reprod Biomed Online. 10 (2), 169-177.

[00102] Damián-Matsumura P, Zaga V, Maldonado A, Sánchez-Hernández C, Timossi C, and Ulloa-Aguirre A. (1999). Oestrogens regulate pituitary alpha2,3-sialyltransferase messenger ribonucleic acid levels in the female rat. J MoI Endocrinol. 23 (2), 153-165.

[00103] D'Antonio M., Borrelli F., Datola A., Bucci R., Mascia M., Polletta P., Piscitelli D., and Papoian R. (1999) Biological characterization of recombinant human follicle stimulating hormone isoforms. Human Reproduction 14, 1160-1167

[00104] Dalpathado DS, lrungu J, Go EP1 Butnev VY, Norton K, Bousfield GR, and Desaire H. (2006). Comparative glycomics of the glycoprotein follicle stimulating hormone: glycopeptide analysis of isolates from two mammalian species. Biochemistry. 45 (28), 8665-8673. No copy

[00105] Dias JA, Van Roey P. (2001). Structural biology of human follitropin and its receptor. Arch Med Res. 32 (6), 510-519

[00106] Fiddes, J. C. and Goodman, H. M. (1979) Isolation, cloning and sequence analysis of the cDNA for the alpha-subunit of human chorionic gonadotropin. Nature, 281, 351-356.

[00107] Flack, M.R., Bennet, A.P., Froehlich, J. Anasti, JN and Nisula, B. (1994). Increased biological activity due to basic isoforms in recombinant human follicle-stimulating hormone produced in a human cell line. J. Clin. Endocrinol. Metab., 79, 756-760

[00108] Fox KM, Dias JA, and Van Roey P. (2001). Threedimensional structure of human follicle-stimulating hormone. MoI Endocrinol. 15 (3), 378-89

[00109] Grabenhorst E, Hoffmann A, Nimtz M, Zettlmeissl G, and Conradt HS. (1995). Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal (beta 1-4) GlcNAc-R alpha 2,6- sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal (beta 1-4) GlcNAc ( beta 1-2) Man (alpha 1-3) -branch of diantennary oligosaccharides from secreted recombinant beta-trace protein. Eur J Biochem. 232 (3), 718-25.

[00110] Green ED and Baenziger JU. (1988). Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J Biol Chem. 263 (1). 36-44.

[00111] Grundmann.U., Nerlich.C, Rein.T. and Zettlmeissl, G. (1990). Complete cDNA sequence encoding human beta-galactoside alpha-2,6-sialyltransferase. G Nucleic Acids Res. 18 (3), 667

[00112] Howies, CM. (1996). Genetic engineering of human FSH (Gonal-F). Hum Reprod. Update, 2,172-191.

[00113] Kagawa Y1 Takasaki S, Utsumi J, Hosoi K, Shimizu H, Kochibe N, and Kobata A. (1988). Comparative study of the asparagine- linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells. J Biol Chem. 263 (33), 17508-17515.

[00114] Keene, J. L., Matzuk, M. M., Otani, T., Fauser, B1C1J1M., Galway, A.B., Hsueh, A.J.W. and Boime, I. (1989). Expression of Biologically active Human Follitropin in Chinese Hamster Ovary Cells. The Journal of Biological Chemistry, 264 (9), 4769-4775.

[00115] Kitagawa.H. and Paulson.J.C (1994) Cloning of a novel alpha 2,3-sialyltransferase that sialylates glycoprotein and glycolipid carbohydrate groups. J. Biol. Chem. 269 (2), 1394-1401.

[00116] Lee EU, Roth J, and Paulson JC (1989) Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6-sialyltransferase. J Biol Chem. 264 (23), 13848-13855.

[00117] by Leeuw, R., Mulders, J., Voortman, G. Rombout, F.Damm, J. and Kloosterboer, L. (1996) Structure-function relationship of recombinant follicle stimulating hormone (Puregon). MoI. Hum. Reprod., 2, 361-369.

[00118] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193 (1). 265-75.

[00119] Lowry, PJ, McLean, C, Jones RL and Satgunasingam N. (1976) Purification of anterior pituitary and hypothalamic hormones Clin Pathol Suppl (Assoc Clin Pathol). 7, 16-21.

[00120] Pierce JG, and Parsons TF (1981) Glycoprotein hormones: structure and function Annu Rev Biochem. 50, 465-495.

[00121] Pricer WE Jr, and Ashwell G. (1971). The binding of desialylated glycoproteins by plasma membranes of rat liver. J Biol Chem. 246 (15), 4825-33.

[00122] Rathnam P, and Saxena BB. (1975). Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit. J Biol Chem .; 250 (17): 6735-6746.

[00123] Regoeczi E, Debanne MT, Hatton MC, and Koj A. (1978) Elimination of asialofetuin and asialoorosomucoid by the intact rat. Quantitative aspects of the hepatic clearance mechanism. Biochim Biophys Acta. 541 (3), 372-84.

[00124] Royle L, Radcliffe CM, Dwek RA and Rudd PM (2006) Methods in Molecular Biology, ed I Brockhausen-Schutzbach (Humana Press), 347: Glycobiology protocols, 125-144.

[00125] Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO and Vutyavanich T. (1987). Structure-function relationships of gonadotropins. Recent Prog Horm Res .; 43: 383-429.

[00126] Saxena BB and Rathnam P. (1976) Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands. J Biol Chem. 251 (4). 993-1005

[00127] Steelman SL, and Pohley FM. (1953) Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology. 53 (6), 604-616.

[00128] Steer CJ, and Ashwell G. (1980) Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes. J Biol Chem. 255 (7), 3008-13.

[00129] Svensson EC, Soreghan B, and Paulson JC. (1990) Organization of the beta-galactoside alpha 2,6-sialyltransferase gene. Evidence for the transcriptional regulation of terminal glycosylation. J Biol Chem. 265 (34): 20863-20868.

[00130] Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, and Kobata A (1988). Comparative study of the asparagine- linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Biol Chem. 263 (8), 3657-3663.

[00131] Timossi CM, Barrios de Tomasi J, Zambrano E, Gonzalez R, Ulloa-Aguirre A. (1998). A naturally occurring basically charged human follicle-stimulating hormone (FSH) variant inhibits FSH-induced androgen aromatization and tissue-type plasminogen activator enzyme activity in vitro. Neuroendocrinology. 67 (3), 153-163.

[00132] Timossi CM, Barrios-de-Tomasi J, Gonzalez-Suarez R, Arancz MC, Padmanabhan V, Conn PM, and Ulloa-Aguirre A. (2000). Differential effects of the charge variants of human follicle-stimulating hormone. J Endocrinol. 165 (2), 193-205.

[00133] Ulloa-Aguirre, A., Espinoza, R., Damian-Matsumura, P. and Chappel, S. C. (1988) Immunological and biological potencies of the different molecular species of gonadotrophins. Hum. Reprod. 3, 491501.

[00134] Ulloa-Aguirre, A., Cravioto, A., Damian-Matsumura, P. Jimenez, M, Zambrano, E and Diaz-Sanchez, V. (1992) Biological characterization of the naturally occurring analogues of intrapituitary human follicle stimulating hormone. Hum. Reprod. 7, 23-30.

[00135] Ulloa-Aguirre A, Midgley AR Jr, Beitins IZ, and Padmanabhan V. (1995). Follicle-stimulating isohormones: characterization and physiological relevance. Endocr Rev. 16 (6), 765-787.

[00136] Ulloa-Aguirre A, Maldonado A, Damian-Matsumura P, and Timossi C (2001). Endocrine regulation of gonadotropin glycosylation. Arch Med Res. 32 (6), 520-532.

[00137] Ulloa-Aguirre A, Timossi C, Barrios-de-Tomasi J, Maldonado A, and Nayudu P. (2003). Impact of carbohydrate heterogeneity in function of follicle-stimulating hormone: studies derived from in vitro and in vivo models. Reprod Biol. 69 (2), 379-389.

[00138] Van Lenten L, and Ashwell G. (1972) The binding of desialylated glycoproteins by plasma membranes of rat liver. Development of a quantitative inhibition assay. J Biol Chem. 247 (14), 4633-40.

[00139] Wide, L. and Albertsson-Wikland, K. (1990) Change in electrophoretic mobility of human follicle-stimulating hormone in serum after administration of gonadotropin-releasing hormone. J. Clin. Endocrinol. Metab. 70, 271-276.

[00140] Wide, L. and Bakos, O. (1993). More basic forms of both human follicle- stimulating hormone and luteinizing hormone in serum at midcycle compared with the follicular or luteal phase. J. Clin. Endocrinol. Metab., 76, 885-889.

[00141] Wide L, Naessen T, Sundstrom-Poromaa I, Eriksson K. (2007) Sulfonation and sialilation of gonadotropins in women during the menstrual cycle, after menopause, and with polycystic ovarian syndrome and in men. J Clin Endocrinol Metab .; 92 (11). 4410-4417.

[00142] Zambrano E, Zariqan T, Olivares A, Barrios-de-Tomasi J, and Ulloa- Aguirre A. (1999). Receptor binding activity and in vitro biological activity of the human FSH charge isoforms as disclosed by heterologous and homologous assay systems: implications for the structure-function relationship of the FSH variants. Endocrine. 10 (2). 113121.

[00143] Zhang X, Lok SH, and Kon OL (1998) Stable expression of human alfa-2,6-sialyltransferase in Chinese hamster ovary cells: functional consequences for human erythropoietin expression and bioactivity. Biochim Biophys Acta. 1425 (3). 441-452.

Claims (7)

1. Recombinant follicle stimulating hormone (rFSH), characterized by the fact that it includes α2,3- and a2,6-sialylation, in which 80% or more of the total sialylation is a2,3-sialylation and in which 5% 20% of total sialylation is α2,6-sialylation. 2. Recombinant follicle stimulating hormone (rFSH), according to claim 1, characterized by the fact that it is expressed in a human cell line. 3. Pharmaceutical composition, characterized by the fact that it comprises rFSH as defined in claim 1 or 2. Pharmaceutical composition according to claim 3, characterized by the fact that it also comprises hCG and / or LH. 5. Pharmaceutical composition according to claim 3 or 4, characterized by the fact that it is for use in the treatment of infertility. 6. Production method of rFSH as defined in claim 1 or 2, characterized by the fact that it comprises the stage of production or expression of rFSH in a human cell line, in which the cell line has been modified by the use of α2,3 -sialyltransferase. 7. Use of rFSH as defined in claim 1 or 2, characterized by the fact that it is in the manufacture of a medication for the treatment of infertility.

Images